Polynucleotides encoding an undercaprenyl diphosphate synthase of staphylococcus aureus

ABSTRACT

The invention provides ups polypedes and polynucleotides encoding ups polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ups polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the ups (undecaprenyldiphosphate synthase) family, as well as their variants, herein referredto as “ups,” “ups polynucleotide(s),” and “ups polypeptide(s)” as thecase may be.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up amedically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past few decades. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains that are resistant to some or all of thestandard antibiotics. This phenomenon has created an unmet medical needand demand for new anti-microbial agents, vaccines, drug screeningmethods, and diagnostic tests for this organism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the ups embodiments of the invention, that have a present benefit of,among other things, being useful to screen compounds for antimicrobialactivity. Such factors are also useful to determine their role inpathogenesis of infection, dysfunction and disease. There is also a needfor identification and characterization of such factors and theirantagonists and agonists to find ways to prevent, ameliorate or correctsuch infection, dysfunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to ups, in particular ups polypeptides andups polynucleotides, recombinant materials and methods for theirproduction. In another aspect, the invention relates to methods forusing such polypeptides and polynucleotides, including treatment ofmicrobial diseases, amongst others. In a further aspect, the inventionrelates to methods for identifying agonists and antagonists using thematerials provided by the invention, and for treating microbialinfections and conditions associated with such infections with theidentified agonist or antagonist compounds. In a still further aspect,the invention relates to diagnostic assays for detecting diseasesassociated with microbial infections and conditions associated with suchinfections, such as assays for detecting ups expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to ups polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a ups of Staphylococcus aureus,that is related by amino acid sequence homology to Micrococcus luteusUPS polypeptide. The invention relates especially to ups having anucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:1and SEQ ID NO:2 respectively. Note that sequences recited in theSequence Listing below as “DNA” represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides.

TABLE 1 Ups Polynucleotide and Polypeptide Sequences (A) Staphylococcusaureus ups polynucleotide sequence [SEQ ID NO:1].5′-ATGTTTAAAAAGCTAATAAATAAAAAGAACACTATAAATAATTATAATGAAGAATTAGACTCGTCTAATATACCTGAACATATCGCTATTATTATGGATGGTAATGGGCGATGGGCTAAGAAGCGAAAAATGCCTAGAATTAAAGGCCATTACGAAGGTATGCAAACAATAAAAAAAATTACTAGGATAGCTAGTGATATTGGTGTTAAGTACTTAACTTTATATGCCTTTTCCACTGAAAATTGGTCAAGACCTGAAAGTGAAGTAAATTATATTATGAATTTGCCTGTCAATTTCTTAAAGACATTCTTACCGGAACTAATTGAAAAAAATGTCAAAGTTGAAACAATTGGATTTACTGATAAGTTGCCAAAATCAACGATAGAAGCAATTAATAATGCTAAAGAAAAGACAGCTAATAATACCGGCTTAAAATTAATATTTGCAATTAATTATGGTGGCAGAGCAGAACTTGTTCATAGTATTAAAAATATGTTTGACGAGCTTCATCAACAAGGTTTAAATAGTGATATCATAGATGAAACATATATAAACAATCATTTAATGACAAAAGACTATCCTGATCCAGAGTTGTTAATTCGTACTTCAGGAGAACAAAGAATAAGTAATTTCTTGATTTGGCAAGTTTCGTATAGTGAATTTATCTTTAATCAAAAATTATGGCCTGACTTTGACGAAGATGAATTAATTAAATGTATAAAAATTTATCAGTCACGTCAAAGACGCTTTGGCGGATTGAGTGAGGAGTAG-3′ (B)Staphylococcus aureus ups polypeptide sequence deduced from apolynucleotide sequence in this table [SEQ ID NO:2].NH₂-MFKKLINKKNTINNYNEELDSSNIPEHIAIIMDGNGRWAKKRKMPRIKGHYEGMQTIKKITRIASDIGVKYLTLYAFSTENWSRPESEVNYIMNLPVNFLKTFLPELIEKNVKVETIGFTDKLPKSTIEAINNAKEKTANNTGLKLIFAINYGGRAELVHSIKNMFDELHQQGLNSDIIDETYINNHLMTKDYPDPELLIRTSGEQRISNFLIWQVSYSEFIFNQKLWPDFDEDELIKCIKIYQSRQRRFGGLSEE-COOH

Deposited materials

A deposit comprising a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit . . . TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain comprises a fill length ups gene. The sequence ofthe polynucleotides comprised in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStaphylococcus aureus WCUH 29 strain, which polypeptide is comprised inthe deposited strain. Further provided by the invention are upspolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare ups polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

Ups polypeptide of the invention is substantially phylogeneticallyrelated to other proteins of the ups (undecaprenyl diphosphate synthase)family.

In one aspect of the invention there are provided polypeptides ofStaphylococcus aureus referred to herein as “ups” and “ups polypeptides”as well as biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of ups polypeptide encoded by naturally occurring alleles of aups gene.

The present invention further provides for an isolated polypeptide that:(a) comprises or consists of an amino acid sequence that has at least95% identity, most preferably at least 97-99% or exact identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2; (b) a polypeptideencoded by an isolated polynucleotide comprising or consisting of apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1; (c) a polypeptide encoded by an isolatedpolynucleotide comprising or consisting of a polynucleotide sequenceencode a polypeptide that has at least 95% identity, even morepreferably at least 97-99% or exact identity, to the amino acid sequenceof SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that has a biological activity of ups,and also those that have at least 95% identity to a polypeptide of Table1 [SEQ ID NO:2] and also include portions of such polypeptides with suchportion of the polypeptide generally comprising at least 30 amino acidsand more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

X—(R₁)m—(R₂)—(R₃)_(n) 13 Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStaphylococcus aureus, however, it may preferably be obtained from otherorganisms of the same taxonomic genus. A polypeptide of the inventionmay also be obtained, for example, from organisms of the same taxonomicfamily or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with ups polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Staphylococcus aureus, are also preferred. Furtherpreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeups polypeptides, particularly polynucleotides that encode a polypeptideherein designated ups.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ups polypeptides comprising asequence set out in Table 1 [SEQ ID NO:1] that includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Staphylococcus aureus.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing ups polypeptides andpolynucleotides, particularly Staphylococcus aureus ups polypeptides andpolynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genonic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a ups polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is aups polypeptide from Staphylococcus aureus comprising or consisting ofan amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding ups polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 inE.coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in Table 1 [SEQ ID NO: 1] was discovered in a DNAlibrary derived from Staphylococcus aureus WCUH 29.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 769 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence that has at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1; (b) a polynucleotide sequence encoding a polypeptide that has atleast 95% identity, even more preferably at least 97-99% or 100% exact,to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Staphylococcusaureus, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof, andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gertz et al, Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purify polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 769 set forth in SEQ ID NO:1 ofTable 1, both of that encode a ups polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

X—(R₁)m—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5′ end nucleic acid residue is at the left,bound to R₁, and its 3′ end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Staphylococcus aureus, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polynucleotide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ups havingan amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganiztion) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefill-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingups variants, that have the amino acid sequence of ups polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, modified, deleted and/oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of ups polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nucleicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

Further preferred embodiments of the invention are polynucleotides thatare at least 95% or 97% identical over their entire length to apolynucleotide encoding ups polypeptide having an amino acid sequenceset out in Table 1 [SEQ ID NO:2] and polynucleotides that arecomplementary to such polynucleotides. Most highly preferred arepolynucleofides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to ups polynucleotide sequences, such as thosepolynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 OmM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1 ×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding ups and to isolate cDNAand genomic clones of other genes that have a high identity,particularly high sequence identity, to a ups gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have lee than 30 nucleotide residues or basepairs.

A coding region of a ups gene may be isolated by screening using a DNAsequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to detenine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS: 1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-termnal aminoacids, or amino acids interior to a mature polypeptide (when a matureform has more than one polypeptide chain, for instance). Such sequencesmay play a role in processing of a protein from precursor to a matureform, may allow protein transport, may lengthen or shorten proteinhalf-life or may facilitate manipulation of a protein for assay orproduction, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an activated form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for activity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

In sum, a polynucleotide of the invention may encode a mature protein amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-Dexran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E.coli,streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcusaureus ; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-erived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaiyotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extacellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including anmoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of ups polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof ups polynucleotides and/or polypeptides in a eukaryote, particularlya mammal, and especially a human, will provide a diagnostic method fordiagnosis of disease, staging of disease or response of an infectiousorganism to drugs. Eukaryotes, particularly mammals, and especiallyhumans, particularly those infected or suspected to be infected with anorganism comprising the ups gene or protein, may be deteted at thenucleic acid or amino acid level by a variety of well known techniquesas well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genoricDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizng amplified DNA to labeled ups polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et aL, Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingups nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al, Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit that comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisns,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding ups polypeptide can be used to identify andanalyze mutations. The invention further provides these primers with 1,2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end. Theseprimers may be used for, among other things, amplifying ups DNA and/orRNA isolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetecd and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStaphylococcus aureus, comprising determining from a sample derived froman individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a ups polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of ups polypeptide compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a upspolypeptide, in a sample derived from a host, such as a bodily material,are well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis,antibody sandwich assays, antibody detection and ELISA assays.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libranes, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g.,inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g, inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofups polypeptides and polynucleotides; or may be structural or finctionalmimetics thereof (see Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists, in the absence of an agonist or antagonist, by testing whetherthe candidate compound results in inhibition of activation of thepolypeptide or polynucleotide, as the case may be. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring ups polypeptideand/or polynucleotide activity in the mixture, and comparing the upspolypeptide and/or polynucleotide activity of the mixture to a standard.Fusion proteins, such as those made from Fc portion and ups polypeptide,as herein described, can also be used for high-throughput screeningassays to identify antagonists of the polypeptide of the presentinvention, as well as of phylogenetically and and/or functionallyrelated polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58(1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose that enhance (agonist) or block (antagonist) the action of upspolypeptides or polynucleotides, particularly those compounds that arebacteristatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising ups polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a ups agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the ups polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of ups polypeptide aremost likely to be good anagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in ups polynucleotide or polypeptideactivity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tunbling rate. Proteincomplexes, such as formed by ups polypeptide associating with anotherups polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of ups polypeptide dimers,trimers, tetramers or higher order structures, or structures formed byups polypeptide bound to another polypeptide. Ups polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on ups polypeptide self-association as well as an associationof ups polypeptide and another polypeptide or small molecule. Upspolypeptide can be coupled to a sensor chip at low site density suchthat covalently bound molecules will be monomeric. Solution protein canthen passed over the ups polypeptide -coated surface and specificbinding can be detected in real-time by monitoring the change inresonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for ups polypeptideself-association as well as an association of ups polypeptide andanother polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of ups polypeptide with another upspolypeptide or a different polypeptide ups polypeptide can be coupled toa scintillation-filled bead. Addition of radio-labeled ups polypeptideresults in binding where the radioactive source molecule is in closeproximity to the scintillation fluid. Thus, signal is emitted upon upspolypeptide binding and compounds that prevent ups polypeptideself-association or an association of ups polypeptide and anotherpolypeptide or small molecule will diminish signal.

In other embodiments of the invention there are provided methods foridenting compounds that bind to or otherwise interact with and inhibitor activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detetable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound, and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for ups agonists is a competitive assay thatcombines ups and a potenial agonist with ups-binding molecules,recombinant ups binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. ups can be labeled, such as byradioactivity or a colorimetric compound, such that the number of upsmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist or antagonist of thepolypeptide and/or polynucleotide, by: (a) determining in the firstinstance the three-dimensional structure of the polypeptide and/orpolynucleotide, or complexes thereof, (b) deducing the three-dimensionalstructure for the likely reactive site(s), binding site(s) or motif(s)of an agonist or antagonist; (c) synthesizing candidate compounds thatare predicted to bind to or react with the deduced binding site(s),reactive site(s), and/or motif(s); and (d) testing whether the candidatecompounds are indeed agonists or antagonists. It will be furtherappreciated that this will normally be an iterative process, and thisiterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of ups polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagoni) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the upspolypeptide and/or polypeptide.

In still another approach, expression of the gene encoding endogenousups polypeptide can be inhibited using expression blocking techniques.This blocking may be targeted against any step in gene expression, butis preferably targeted against transcription and/or translation. Anexamples of a known technique of this sort involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., NucleicAcids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial ups proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided ups agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein “H. pylori”) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofups polypeptides and/or polynucleotides) found using screens provided bythe invention, or known in the art, particularly narrow-spectnumantibiotics, should be useful in the treatment of H. pylori infection.Such treatment should decrease the advent of H. pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney andurinarytract (e.g., epididymitis, intrarenaland perinephric absces, toxic shock syndrome), skin (e.g., impetigo,folliculitis, cutaneous abscesses, cellulitis, wound infection,bacterial myositis) bone and joint (e.g., septic arhritis,osteomyelitis).

“Host cell(s)” is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serranaliquefaciens, Vibrio cholera, Shigella dysenteri, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfingens, Clostridiumtetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsiiand Chlamydia trachomitis, (ii) an archaeon, including but not limitedto Archaebacter, and (iii) a unicellular or filamentous eukaryote,including but not limited to, a protozoan, a fungus, a member of thegenus Saccharomyces, Kluveromyces, or Candida, and a member of thespecies Saccharomyces ceriviseae, Kluveromyces lactis, or Candidaalbicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without lirnitation, single-and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commnonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-lining,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylatian, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS-STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Snthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones comprising overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

2 1 771 DNA Staphylococcus aureus 1 atgtttaaaa agctaataaa taaaaagaacactataaata attataatga agaattagac 60 tcgtctaata tacctgaaca tatcgctattattatggatg gtaatgggcg atgggctaag 120 aagcgaaaaa tgcctagaat taaaggccattacgaaggta tgcaaacaat aaaaaaaatt 180 actaggatag ctagtgatat tggtgttaagtacttaactt tatatgcctt ttccactgaa 240 aattggtcaa gacctgaaag tgaagtaaattatattatga atttgcctgt caatttctta 300 aagacattct taccggaact aattgaaaaaaatgtcaaag ttgaaacaat tggatttact 360 gataagttgc caaaatcaac gatagaagcaattaataatg ctaaagaaaa gacagctaat 420 aataccggct taaaattaat atttgcaattaattatggtg gcagagcaga acttgttcat 480 agtattaaaa atatgtttga cgagcttcatcaacaaggtt taaatagtga tatcatagat 540 gaaacatata taaacaatca tttaatgacaaaagactatc ctgatccaga gttgttaatt 600 cgtacttcag gagaacaaag aataagtaatttcttgattt ggcaagtttc gtatagtgaa 660 tttatcttta atcaaaaatt atggcctgactttgacgaag atgaattaat taaatgtata 720 aaaatttatc agtcacgtca aagacgctttggcggattga gtgaggagta g 771 2 256 PRT Staphylococcus aureus 2 Met PheLys Lys Leu Ile Asn Lys Lys Asn Thr Ile Asn Asn Tyr Asn 1 5 10 15 GluGlu Leu Asp Ser Ser Asn Ile Pro Glu His Ile Ala Ile Ile Met 20 25 30 AspGly Asn Gly Arg Trp Ala Lys Lys Arg Lys Met Pro Arg Ile Lys 35 40 45 GlyHis Tyr Glu Gly Met Gln Thr Ile Lys Lys Ile Thr Arg Ile Ala 50 55 60 SerAsp Ile Gly Val Lys Tyr Leu Thr Leu Tyr Ala Phe Ser Thr Glu 65 70 75 80Asn Trp Ser Arg Pro Glu Ser Glu Val Asn Tyr Ile Met Asn Leu Pro 85 90 95Val Asn Phe Leu Lys Thr Phe Leu Pro Glu Leu Ile Glu Lys Asn Val 100 105110 Lys Val Glu Thr Ile Gly Phe Thr Asp Lys Leu Pro Lys Ser Thr Ile 115120 125 Glu Ala Ile Asn Asn Ala Lys Glu Lys Thr Ala Asn Asn Thr Gly Leu130 135 140 Lys Leu Ile Phe Ala Ile Asn Tyr Gly Gly Arg Ala Glu Leu ValHis 145 150 155 160 Ser Ile Lys Asn Met Phe Asp Glu Leu His Gln Gln GlyLeu Asn Ser 165 170 175 Asp Ile Ile Asp Glu Thr Tyr Ile Asn Asn His LeuMet Thr Lys Asp 180 185 190 Tyr Pro Asp Pro Glu Leu Leu Ile Arg Thr SerGly Glu Gln Arg Ile 195 200 205 Ser Asn Phe Leu Ile Trp Gln Val Ser TyrSer Glu Phe Ile Phe Asn 210 215 220 Gln Lys Leu Trp Pro Asp Phe Asp GluAsp Glu Leu Ile Lys Cys Ile 225 230 235 240 Lys Ile Tyr Gln Ser Arg GlnArg Arg Phe Gly Gly Leu Ser Glu Glu 245 250 255

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide sequence or the full complement of the entire length ofthe polynucleotide sequence, wherein the polynucleotide sequence isidentical to SEQ ID NO:1, except that, over the entire lengthcorresponding to SEQ ID NO:1, n_(n) nucleotides are substituted,inserted or deleted, wherein n_(n) satisfies the following expression n_(n) ≦x _(n)−(x _(n) ·y) wherein x_(n) is the total number ofnucleotides in SEQ ID NO:1, y is at least 0.95, and wherein anynon-integer product of x_(n) and y is rounded down to the nearestinteger before subtracting the product from x_(n); and wherein thepolynucleotide sequence detects Staphylococcus aureus by hybridizationand wherein the polynucleotide sequence is not genomic DNA.
 2. A vectorcomprising the isolated polynucleotide of claim
 1. 3. An isolated hostcell comprising the vector of claim
 2. 4. The isolated polynucleotide ofclaim 1, wherein y is at least 0.97.
 5. The isolated polynucleotide ofclaim 1, wherein y is at least 0.99.
 6. An isolated polynucleotidecomprising a polynucleotide sequence or the full complement of theentire length of the polynucleotide sequence, wherein the polynucleotidesequence comprises SEQ ID NO:1: wherein the polynucleotide sequencedetects Staphylococcus aureus by hybridization and wherein thepolynucleotide sequence is not genomic DNA.
 7. A vector comprising theisolated polynucleotide of claim
 6. 8. An isolated host cell comprisingthe vector of claim
 7. 9. A process for producing a polypeptidecomprising culturing the host cell of claim 8 under conditionssufficient for the production of the polypeptide, wherein thepolypeptide comprises SEQ ID NO:2.
 10. The isolated polynucleotide ofclaim 6 encoding a fusion polypeptide, wherein the fusion polypeptidecomprises SEQ ID NO:2.
 11. An isolated polynucleotide comprising apolynucleotide sequence or the full complement of the entire length ofthe polynucleotide sequence, wherein the polynucleotide sequencehybridizes to the full complement of SEQ ID NO:1, wherein thehybridization conditions include incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA,followed by washing in 0.1×SSC at 65° C.; and, wherein thepolynucleotide sequence is identical to SEQ ID NO:1, except that, overthe entire length corresponding to SEQ ID NO:1, nn nucleotides aresubstituted, inserted or deleted, wherein n_(n) satisfies the followingexpression n _(n) ≦x _(n)−(x _(n) ·y) wherein x_(n) is the total numberof nucleotides in SEQ ID NO:1, y is at least 0.95, and wherein anynon-integer product of x_(n) and y is rounded down to the nearestinteger before subtracting the product from x_(n); and wherein thepolynucleotide sequence detects Staphylococcus aureus by hybridizationand wherein the polynucleotide sequence is not genomic DNA.
 12. Theisolated polynucleotide of claim 11, wherein y is at least 0.97.
 13. Anisolated polynucleotide comprising a polynucleotide sequence, whereinthe polynucleotide encodes a polypeptide comprising SEQ ID NO:2, whereinthe polynucleotide sequence is not genomic DNA.
 14. A vector comprisingthe isolated polynucleotide segment of claim
 13. 15. An isolated hostcell comprising the vector of claim
 14. 16. A process for producing apolypeptide comprising culturing the host cell of claim 15 underconditions sufficient for the production of the polypeptide, wherein thepolypeptide comprises SEQ ID NO:2.
 17. The isolated polynucleotide ofclaim 13 encoding a fusion polypeptide, wherein the fusion polypeptidecomprises SEQ ID NO:2.
 18. An isolated polynucleotide comprising apolynucleotide sequence, wherein the polynucleotide encodes apolypeptide consisting of SEQ ID NO:2; wherein the polynucleotidesequence is not genomic DNA.
 19. A vector comprising the isolatedpolynucleotide of claim
 18. 20. An isolated host cell comprising thevector of claim
 19. 21. A process for producing a polypeptide comprisingculturing the host cell of claim 20 under conditions sufficient for theproduction of the polypeptide, wherein the polypeptide consists of SEQID NO:2.